JPS6284099A - Novel production of peptide - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain a peptide useful as an intermediate for sweeteners, etc., by reacting an alpha-amino acid with a trialkylsilylimidazole and reacting the resultant compound with an active ester, e.g. an alpha-amino acid pyridine thioester having protected amino group in an organic solvent. CONSTITUTION:An alpha-amino acid having formula I [R<5> is H, 1-10C alkyl, 2-6C alkenyl, phenyl or 1-6C alkyl substituted by (protected)hydroxyl group, etc.] is reacted with a trialkylsilylimidazole and the resultant compound is then reacted with an active ester compound having formula II [R<6> is H, 1-10C alkyl 2-6C alkenyl, phenyl, 1-6C alkyl substituted by protected hydroxyl group; R<7> is protecting group of amino group or group expressed by formula III (R<8> is protecting group of amino group)] in an organic solvent or deprotected or dealkylated after the reaction to afford the aimed compound ecpressed by formula IV (R<1> is group of the same kind as R<5>; R<2> is a group of the same kind as R<7>; R<3> is H or lower alkyl).

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to the general formula (1), where R1 is a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, a phenyl group, Carbon number 7-9
an aralkyl group having 1 to 6 carbon atoms substituted with a protected or unprotected hydroxyl group, an aralkyl group having 7 to 9 carbon atoms substituted with a protected or unprotected hydroxyl group, a lower alkylthio group substituted with an alkyl group having 2 to 6 carbon atoms, a 1-carbon alkyl group substituted with a protected or unprotected thiol group
~4 alkyl group represents an indolylmethyl group, R2 is a hydrogen atom, a protecting group for an amine group (herein R
1 represents the same meaning as above, R4 represents a hydrogen atom or a protecting group for an amine group), and R3 represents a hydrogen atom or a lower alkyl group.

More specifically, the present invention is an extremely useful method for synthesizing R peptides without decomposition of active esters in organic solvents or racemization with bases, and is also a breakthrough in that α-amino acids can be used without protecting groups. This is a typical method.

The compounds obtained by the method of the present invention are useful as various hormones, agricultural chemicals, foods (e.g., sweeteners), or as intermediates for synthesizing final target compounds having various uses as described above from the compounds.

(Prior Art) The synthesis of iptides is broadly divided into solid phase synthesis and liquid phase synthesis. The solid-phase method has the advantage of requiring fewer purification steps, allowing rapid synthesis, and saving labor by using a synthesis machine. On the other hand, the liquid phase method is generally superior in yield and 2-semization ratio in each step, and both methods have advantages.

In the liquid phase method, the basic synthesis methods are classified into three. (1) C-terminal activation method (2) Condensation method (3) N-terminal activation method (isocyanate method) In method (1), the carboxyl group of one amino acid is used as an active ester, and the other amino acid and a base are This is a method of condensation in the presence or absence of the amino acid, and method (2) uses a condensing agent to convert the carboxyl group of one amino acid into an active ester in a syringe flask, and converts it into an active ester with the amine group of the other amino acid. This is a condensation method.

These two methods are most commonly used in liquid phase synthesis of various peptides.

However, in both cases, it is usually essential to protect the carboxyl group of the amine component condensed to the active ester by esterification.

Amino acids are naturally neutralized by forming dissociated inner salts. Therefore, it is poorly soluble in water and organic solvents.

For this reason, in the liquid phase method, the carboxyl group must be protected and neutralized in order to bring out the basicity (nucleophilicity) of the nitrogen of the amino group.

One way to bring out the basicity of the amine in an amino acid is to add a strong base to form a carboxylate. This method is proposed by Schotten-Bauman (Schotten-B.
The Bodanski (B. aurnan) method is known.
odansky) La < Peptide Synthesis 2nd J + Edition (Peptide 5ynthes)
However, in this method, the active ester group undergoes a competitive reaction between the amino group and the hydrolysis in water, making it unstable (
It is not possible to use active esters with high activity.

(Problems to be Solved by the Invention) The purpose of the present invention is to carry out the reaction in an organic solvent in order to prevent the decomposition of the active ester by water, and to perform the reaction under near-neutral reaction conditions in order to prevent racemization by the base. This is to bring out the basicity of the amine.

Another object of the present invention is to provide a method that satisfies the above conditions without protecting α-amino acids in order to further expand the range of applications.

(Means for Solving the Problems) As a method for neutralizing the carboxyl groups of free amino acids, bringing out the basicity of the amino groups, and solubilizing them in organic solvents, the present inventors have investigated a method using silylation.

The silylation was solved by using trimethylsilylimidazole.

α-Amino acid and trimethylsilylimidazole at 0℃~
When the reaction is carried out in an organic solvent at 30°C, the reaction proceeds quickly and N
-) trimethylsilylaminotrimethylsilyl ester is obtained.

Taking advantage of this, the general formula (2) (wherein R5 is a hydrogen atom, an alkyl group having 1 to 10 carbon atoms,
Alkenyl group having 2 to 6 carbon atoms, phenyl group, 7 to 6 carbon atoms
9 aralkyl group, 1 alkyl group having 1 to 6 carbon atoms substituted with a protected or unprotected hydroxyl group, 1 aralkyl group having 7 to 9 carbon atoms substituted with a protected or unprotected hydroxyl group,
Alkyl group having 2 to 6 carbon atoms substituted with lower alkylthio group, 1 to 4 carbon atoms substituted with protected thiol group
When an α-amino acid having an alkyl group or an indolylmethyl group is treated with trimethylsilylimidazole in an organic solvent, it has the general formula (4) (where A is a hydrogen atom,
Alkyl group having 1 to 10 carbon atoms, alkenyl group having 2 to 6 carbon atoms, phenyl group, aralkyl group having 7 to 9 carbon atoms, alkyl group having 1 to 6 carbon atoms substituted with a protected hydroxyl group, protected an aralkyl group having 7 to 9 carbon atoms substituted with a hydroxyl group; an aralkyl group having 2 to 9 carbon atoms substituted with a lower alkylthio group;
6 alkyl group, an alkyl group having 1 to 4 carbon atoms substituted with a protected thiol group, or an indolylmethyl group) is obtained.

In this step, the reaction temperature is preferably 00 to 30°C, and the reaction solvent is preferably amide, ether, ester, or chlorine. The amount of trimethylsilylimidazole used for the α-amino acid may be an amount that provides the general formula (4).

N-trimethyl using trimethylsilylimidazole
The advantage of using nlylaminotrimethylsilyl ester is that the imidazole that appears in the reaction system is a neutral compound that does not take part in the reaction and there is no fear of racemization.

This means that without isolating the N-trimethylsilylamino trimethylsilyl ester, an active ester can be added in the following steps to synthesize peptide.

Regarding the α-amino acid group represented by the general formula (2), it is best to have an equivalent amount with respect to the amine group and the carboxyl group, that is, 2 equivalents, but even if it is 1 equivalent, the peptide-forming reaction can be prevented by using the following reaction conditions. proceed. However, it is inevitable that the yield will decrease when the amount is 1 equivalent.

Next, a condensation reaction between N-)limethylsilylaminotrimethylsilyl ester having the general formula (4) and an active ester compound of an amino acid was investigated.

The α-amino acid of the general formula (2) is reacted with trimethylsilylimidazole to form a compound having the general formula (4), and without isolation, the α-amino acid of the general formula (3) [wherein R6 is a hydrogen atom and has 1 to 10 carbon atoms] an alkyl group having 2 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, a phenyl group, an aralkyl group having 7 to 9 carbon atoms, an alkyl group having 1 to 6 carbon atoms substituted with a protected hydroxyl group, and an alkyl group having 1 to 6 carbon atoms substituted with a protected hydroxyl group. an aralkyl group having 7 to 9 carbon atoms, an alkyl group having 2 to 6 carbon atoms substituted with a lower alkylthio group, an alkyl group having 1 to 4 carbon atoms substituted with a protected thiol group, or an indolylmethyl group. By adding an amino acid active ester compound having the following formula, R7 represents an amine group-protecting group or a group (wherein R6 represents the same meaning as above and R8 represents an amino group-protecting group). The general formula (
1a) A compound of the present invention having the formula (wherein A, R6 and R7 have the same meanings as above) is obtained.

The active esters used in this reaction are p-nitrophenyl ester, dinitrophenyl ester, hantachlorphenyl ester, trichlorphenyl ester nato phenols, N-oxysuccinimide ester,
Although esters of N-oxyphthalimide and the like may be used, pyridylthioesters are preferred.

The above is a novel synthesis method for peptides, first of all, the general formula (
The amino acid of 2) is suspended in an organic solvent, and then trimethylsilylimidazole is added. As the reaction progresses, the insoluble matter dissolves, forming a translucent solution. For example, when dimethylformamide is used as a solvent, a translucent solution is obtained, and the formation of two-trimethylsilyl ester of N-) +) methylsilylua can be easily confirmed.

When an excess of trimethylsilylimidazole is used, one reaction does not proceed. This is thought to be due to the formation of an N,N-u trimethylsilylamino compound having the general formula (5) (wherein A has the same meaning as above). This means that the amount of citrimethylsilylimidazole must always be used accurately.

When the carboxyl group of the above-mentioned compound (la) of the present invention is reacted with a general alkylating agent, the formula (1b) (wherein A,
Other compounds of the present invention are obtained in which R6 and R7 have the same meanings as above, and R9 represents a lower alkyl group.

The alkylating agent used here may be a general alkylating agent, such as diazomethane.

In addition, by removing the protective group of the above-mentioned compound (1a) using a method generally used in petido chemistry, another compound of the present invention (1c) [wherein RIO is a hydrogen atom and has 1 to 10 carbon atoms] Alkyl group with 2 to 6 carbon atoms, phenyl group, aralkyl group with 7 to 9 carbon atoms, alkyl group with 1 to 6 carbon atoms substituted with hydroxyl group, 7 to 9 carbon atoms substituted with hydroxyl group represents an aralkyl group, an alkyl group having 2 to 6 carbon atoms substituted with a lower alkylthio group, an alkyl group having 1 to 4 carbon atoms substituted with a thiol group, or an indolylmethyl group, and R11 is a hydrogen atom or a group. (Here, RIO has the same meaning as above)] The peptide of the present invention can be obtained.

To summarize the production method of the present invention, first, trimethylsilylimidazole is added to an amino acid to produce N-trimethylsilylamino trimethylsilyl ester of the amino acid.

What is separated here is imidazole and does not participate in the reaction at all. Then the active ester (pyridylthioester) is added.

What separates here is 2-pyridinethiol.

Under such reaction conditions, there is no need to add a catalyst such as a weak base. There is also no need to isolate silylated amino acids as intermediates.

The compounds present in the reaction system are almost neutral and there is no fear of racemization. The reaction conditions are preferably room temperature or lower, and since the reaction proceeds at room temperature to -20'C, no temperature control is required.

After completion of the reaction, imidazole and 2-pyridinethiol can be removed by washing with water.

Thereby, after concentration, it can be easily purified by recrystallization, column chromatography, preparative thin layer chromatography, etc.

It is thought that the yield can be determined quantitatively by considering the type of amino acid, solvent, reaction time, etc.

Using the method of the present invention, it is theoretically possible to extend the peptide chain to any length as long as the solvent is compatible.

(Examples) The present invention will be explained in more detail below using Examples and Reference Examples, but it goes without saying that the present invention is not limited to these Examples.

Example 1 N-(benzyloxycarbonyl)-L-valyl-2-
Trimethylsilylimidazole (56(19)) was added to a suspension of L-allylglycine amino-4-pentenoate (23(ly)) in dimethylformamibi (5i/l') under a nitrogen stream at room temperature, and the mixture was stirred for 1 hour.

Next, a dimethylformamibi solution (3 mJ) of N-penzyloxycarbonylvaline hyricyl thioester (550 ml) was added at room temperature and stirred for 2 hours.

The reaction solution was poured into water, adjusted to pH 2 with IN hydrochloric acid, stirred for 30 minutes, and extracted three times with ethyl acetate (200 ml).
The organic layer was washed with saturated brine, dried and concentrated to give 50C crude crystals.
1 g (yield: 88 g) was obtained.

Recrystallized from chloroform/hexane to obtain 467mp (yield: 8
1%) of the title compound was obtained.

In Example 1, it was found that the optimum amount of trimethylsilylimidazole was 2 equivalents based on the following experimental examples.

Further, in order to examine the influence of the solvent, an experiment was conducted using the same method as in Example 1 using the following solvents.

By dissolving the title compound in ether and adding excess diazomethane, N-(benzyloxycarbonyl)-L
-Bav-2-amino-4- was citenic acid methyl ester.

The physical properties of methyl ester are shown below.

Melting point: 116.5-117.0°C [α)D: +12.9° (C-1,01, chloroform) IH chloride (chloroform, cm ): 34
50°1740.1720.1680 7 Su, 2. -<Ktor (m/z): 363 (M
+l f, 254° NMR (CDC13, δ
): 0.87 (3H, d.

J=7.0H2), 0.93 (3H,d, J-7
,0H2).

2.06 (IH, w), 2.48 (2H, t, J-7,
0Hz).

3.68 (3H, s), 4.00 (IH, aa, J-6
゜8.5H2) + 4.60 (I H,, it,
J=6. 8.5H2) r5.04(2H, θ)
, 6.46 (IH,a, J-8,5).

7.28 (5H, θ) Example 2 Add dimethylformamide silylimidazole (176 μl) of N-(-cundyloxycarbonyl)-valyl-L-methionine L-71 thionini (90 ml F) and stir for 2 hours. I saw it.

Next, a dimethylformamide solution (2 ml) of N-<ndyloxycarbonyl-L-par-2-biridylnaonister<17omy) was added and stirred for 14 hours.

The reaction was stopped by adding 3me of IN hydrochloric acid, and after extraction with ethyl acetate,
The organic layer was treated in the same manner as in Example flJ 1 to obtain the title compound 168 (yield: 89%) as white crystals.

Melting point: 180-182.

3α:] D knee 22.6° (C = 1.05, chloroform) IRx-e crtor (nujol, crrL-1
):335o.

3300, 1740, 1640 Mask is kutle (m/z): 276, 203, 183N
M R Sufu) Tor (CD a OD, J
): o. 9 6 (3 H, d.

J=7. OHz), O. 99 (3H, d,
J'=7.0Hz).

2,05 (3H, s), 3.96 (IH,
d, J-8.0Hz).

4, 58 (IH, ad, T=4.8Hz), 5.
08 (2H.

s), 7.32 (5H, B) Example 3 N-(benzyloxycarbonyl)-valyl-L-phenylalanine maple ester L-phenylalanine (99 mL) was sold in dimethylformamide at 1 hour in Shu, Night (1 me ), trimethylsilylimidanol (176 μl) and 2-pyridylthioester of N-benzyloxycarbonyl-L-valine (1
612g) in dimethylformamide solution (1 ml) was reacted and treated in the same manner as in Example 2. The crude crystals obtained were dissolved in 5 mJ of ethyl acetate, and an excess of an ether solution of diazomethane was added to form the methyl ester. did.

After concentration, it was subjected to silica gel column chromatography and eluted with ether/hexane (25 nia 5) to give the title compound as white crystals at 1717 ffp. (yield 86%).

Melting point: 142-143°C [α]D: +366° (C=1.1, chloroform) mass, X. <Rir (m/z): 412 (M+
), 304.

245, 206 IH Su-! 'Chloroform, cm-'':
3450.

3350, 1750, 1740.

1730, 168O
δ): 0.87 (3H.

d. J=60Hz), 093(3H,d,J-6,oH
z).

2, 08 (IH, q, J=6Hz),
3.10 (2H, d.

J = 6Hz), 3.72 (3H, s), j, 00 (IH
, dd.

J=6. 9Hz) + 4.88 (IH, dt, J
-6,13Hzλ5.10(2H,s), 5.34
(IH, d, J-9H2).

6.36 (IH, d, J=8Hz), 7.36 (5H,
e).

7.0-7.4 (5H, m) Example 4 A suspension of N-(benzyloxycarbonyl]valylclidiglycine <1 oomy) in dimethylformamide (1 m
J),) Treated in the same manner as in Example 1 using lyntylsilylimidazole (390 μl) and a dimethylformamide solution (1,0 tnl) of pyridylthioester of N-benzyloxycarbonyl-L-,s-phosphorus (381"?). , 'The reaction was carried out at room temperature for 16 hours.Similarly to Example 1, Treatment I ~ The obtained oil was dissolved in ethyl acetate, and an ether solution of diazomethane was added to obtain a methyl ester.This methyl ester was subjected to silica gel column chromatography. (Elution solvent: ether) to obtain 377m9 of the title compound as white crystals (yield: 97m).

Melting point: I59.5-161.5°C [α) D knee 13.1° (C-1,16, methanol)
Engineering Rs (Chloroform l-cm-”):
3450 r1750.1740,1720°? Susu-<cuttle (m/z): 322 (M”
), 255°NMR vector (CDC13, δ)
:0.94 (3H, d.

J=7Hz), 0.98 (3H,d, J=7H
z), 2.05 (IH, q, J-7H2), 3.72
(3H, S).

3.90-4.22 (3H, m), 5.08 (2H, s
).

5.58 (IH, d, J-9Hz), 6.80 (LH.

dd, J=5.6Hz), 7.32 (5H, 8) Example 5 N-(benzyloxycarbonyl)-L-valyl-L-
Threonine methyl ester ○H To a dimethylformamide suspension (0.5 ml) of L-threonine (238 m9) was added dropwise trimethylsilylimidazole (88 μl) under a nitrogen stream at room temperature and stirred to obtain a translucent solution. Next, N-benzyloxi7carbonyl-L
A dimethylformamide solution of -valine pyridyl thioester (6111!i+) was added dropwise and treated in the same manner as in Example 4. The resulting oil was treated with excess diazomethane ether solution. The crude product was subjected to silica gel column chromatography to obtain the title compound as white crystals. Yield: 295 mg, yield: 41 pieces.

Melting point: 141-142°C [α) D knee 8.3° (C-1,0, chloroform) Mass spectrum (m/z): 366 (M”),
322 Engineering Rs Kutle (Riooholm, cm)
, 3450° 1750.1730, 1710° 68O NMR speed? (CDCl3.δ): 0.9
3 (3H, d.

J=7Hz), Q, 9 et al. (3H, d, J=7H2)
, 1.16 (3Hldr J #7 Hz)
+370 (3H+ days).

4.05 (IH, dd, J=7.9Hz),
4.58 (l H.

d d r J = 2-5 r 8 Hz), 5
,05 (2H,e) + 5.70(IH,a,
J=8Hz), 7.11 (IH,a, J
=9Hz), 7.30(5H,8) Example 6 N-(-1''silyloxycarbonyl)-L-no;lyl-0-(t-butyldimethylsilyl)-L-threonine methyl ester o -t -fludimethyl/lilu L-threonine 3
Suspension of 8 mg of dimethylformamide (0.5 mg) and 49 μl of trimethyl-7lylimidazole (...
N- was added to the transparent 471 obtained by stirring for 30 minutes.
Benzyloxycarbonyl-L-valine pyridylthioester 56Tng in dimethylformamide 9 (0.5m
/) solution was added dropwise and reacted at room temperature for 3 hours.

Add 2rne of 0.5N hydrochloric acid, extract with ethyl acetate, and process in a conventional manner to obtain a crude product containing excess diazomethane.
Ether solution was added to perform esterification. This esterified product was subjected to column chromatography on silica gel, and the title compound 55r was eluted with silica gel ethyl acetate (3:1).
n9 (70% yield) was obtained as an oil.

[α) D: +4.31° (C-1,16, chloroform) Mask is kutle (m/z): 481 (M+1)”+4
23.315 Engineering Rx-? Cuttle (film + cm): 34501
3300.1725,167O NMR spectrum (CDCI 3. δ): 0.0O
(3H.

s), 0.06 (3H, e), 0.85 (9H, s)
.

1.00 (3H, d, J-7H2), 1.03 (3H,
a.

J-7H2), 1.17 (3H, d, J-7
Hz), 2.16(” J q p J-7H
z) t 3.70 (3H+ 8) +4.
13 (IH, ad, J-6,8Hz), 4.
35-4.60 (2H, m), 5.12 (2H, s),
5.50 (IH.

d, J=8H2), 6.36 (IH, d, J-8H2
).

7.35(5H,e) Example 7 N-(t-butocarbonyl)-2-amino-4-,
,2-tenoyl-0-(t-butyldimethylsilyl)-
L-threonine methyl ester NH-COOC (CH3
) 3 O-t-butyldimethylsilylthreonine (953rn
A solution of P (4.0 mmol) in dimethylformamide (8,0 ml) and K)limethylsilylimigsol (1,17 m/8.0 mmol) was added and stirred for 1 hour at room temperature under a nitrogen atmosphere, resulting in a clear solution. became.

To this solution, a methylene chloride solution (10 mA') of N-t-butoxycarbonylallylglycine thioviridyl S fl (1,05,9,3,4 mmol) was slowly added dropwise under stirring at room temperature. After that, stirred overnight at room temperature,
The mixture was poured into water, adjusted to pH 3 with a 10% aqueous citric acid solution, and extracted several times with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a reaction mixture as a yellow oil. (2,25 g) Into an ether solution of a portion of this reaction mixture (4149),
After adding the diazomethane ether solution.

The solution was distilled off, and the resulting colorless oil was subjected to column chromatography to obtain ether/hexane='/1)Nt
-Butoxycarbonylallylglycine-t-butyldimethylsilylthreonine methyl ester was obtained. (2
52my (yield 95%) Properties: Colorless oil IR scratch depth (film, cm-'): 3450°3
335.3080. ．． 1760°1720.1685 7, l! , STR (m/z): 445 (M+l
)+, 400° NMR squictor (CDCI!3.
δ) Knee 0.04 (3H.

s), 0.02 (3H, s), 0.83 (9H, s).

1.14 (3H, d, J-7,0Hz) + 1.42
(9H, s).

2.52 (2H, m), 3.68 (3H, s), 4.1
~4.6 (3H, m), 5.0~5.3 (3H,
m), 5.80 (IH.

d ty J-18,0,10,0,7,0H2)
, 6-67 (I Hldt 1 (10 Hz) Example 8 N-(t-butoxycarbonyl)-2-7 minnow 4- is ntenoyl 0-(t-7' thyldimethylsilyl)-L
Trimethylsilylimidazole (0.65 m/4.4 mmol) was added to a dimethylformamide solution (5 ml) of -threonyl-2-amino-nthenic acid methyl ester allyl glycylate/(255 m9 2.2 mmol) and stirred at room temperature for 1 hour. A clear solution was obtained. To this solution, Nt-ptoxycarbonylallylglycine-0-t
A methylene chloride solution (8 m/m) of -,/tildimethylsilylthreonine thioviridyl ester (966 m/91.85 mmol) was added dropwise at room temperature under a nitrogen stream, followed by stirring overnight. Pour this reaction solution into water and
After adjusting the pH to 3 with % citric acid aqueous solution, the mixture was extracted several times with ethyl acetate. After drying the organic layer with anhydrous magnesium sulfate,
Filtration and distillation of the solvent under reduced pressure gave a yellow oil.

A diazomethane ether solution was added to an ether solution (10 ml) of this product under water cooling at 0°C, and the solvent was distilled off.
The obtained colorless oil was subjected to column chromatography and eluted with (ether/hexane-1/1) to obtain 875 mg (yield: 88%) of the title compound.

Properties: Colorless crystal Melting point = 135 to 1365°C (Recrystallization solvent: hexane)
20゜3380.3080,1740゜1715, 1670 -r Susu-<Kutl(,mlz): 542 (M
+ 1)", 4 g7゜〔α): +9.9°
(C-1,0, chloroform) NMRx-? Cuttle (C
DCl3+ δ): 0.14 (s, 3H).

017 (θ, 3H), 0.93 (9H, θ), 1.1.
2 (3H,a, ,T-6,0H2), 1.45
(9H, s).

2-53 (4H9m) t 3.74 (3H+
e), 419 (IH, a, J-, 6,0Hz
), 4.36 (IH, aa, J=7.0. 3.0
H2), 4.41 (IH, bre), 4.6
5 (IHt (11; I=7.0Hz)1 5.00
(IH, bre).

5, 1-5.2 (4H, m)t 5.6-5.
8 (2H, m,).

7.02 (IH, d, J-6,0Hz), 7.
29 (IH, d.

J-7, oHz).

This compound (Example 8) was 0.5 N hydroxylated) IJ
N-(t-butoxycarbonyl)-2-amino-4--! ! Nthenoyl-0-(t-butyldimethylsilyl)-L-threonyl-
Nthenic acid was obtained in 2-amine.

Properties: Amorphous crystalline NMR spectrum (100MHz CDaODs
δ) 20.08 (3H, θ), 0.10 (θ, 3H
), 0.92 (9H, 8), 1.16 (3H, d, J-
6,0H2).

1.45 (9H, s), 2.52 (4H, m)
, 4.0-6.0 (12H, m), 7.68 (I
H, d, J = 8.0Hz).

7.81 (LH, d, J-8,0Hz).

Example 9 (23,3R)7N-(t-butoxycarbonyl)-2
-amino-3-(tetrahydropyranyl)oxy-4-
(, t-butyldimethylsilyloxyphenyl)-phthyroyl-0-(t-butyldimethylsilyl)-threonine pyridine thioester 0-(1-butyldimethylsilyl)-threonine at 35 mV with 0.5 mV of anhydrous N,N-dimethylformamidine. The suspension was suspended in 3 ml, 44 μl of trimethylsilylimidazole was added, and the mixture was stirred at room temperature for 1 hour under nitrogen.

A solution of Compound 45.5 of Reference Example 3 in anhydrous dimethylformamide (0.7 ml) was then added to the above silylated product at room temperature, and the mixture was further stirred for 15 hours. Water was added to the reaction solution, the pH was adjusted to 44 with IN hydrochloric acid, and the mixture was extracted with ethyl acetate.

The organic layer was dried over anhydrous magnesium sulfate and concentrated to obtain an oil.

This oil was dissolved in 0.5 ml of anhydrous dichloromethane, 23.8 ml of triphenylphosphine and 16.7 ml of dipyridyl disulfide were added, and the mixture was stirred at room temperature under a nitrogen atmosphere.
．． Stirred for 5 hours.

The reaction solution was concentrated under reduced pressure, and the resulting oily substance was subjected to silica gel column chromatography using ether/hexane (
1:1) to give the title compound 38.9 my (yield 63
%) was obtained.

Properties: Oily substance NMR spectrum (CDC13, δ): 0.01.
0.05 (each 3H, day), 0.16 (6H, θ), 0.
88 and 0.90 (8 each. 9H in total), 0.95 (9H)
H, θ), 1.14 and 1.20 (each a + 3 H+
J 6 Hz), 148 (9H+brs),
6.72 and 673 (each a, 2 H, J = 8
Hz) +7.09 (2H9d, J=8 Hz
), 7.16-7.80 (3H, m), 8.59 (
IH, d, J = 5Hz).

Reference Example I N-(t-butoxycarbonyl)-2-amino-4-tintenoinacitubithiopyridyl ester N-t-butoxycarbonylallyl chrysine (500 m
9 2.3 mmol) in methylene chloride solution (8 ml
), dipyridyl sulfide (511+7゜2.3 mmol), then triphenylphosphine (731m?).
, 2.8 mmol) were added. After stirring overnight at room temperature, the solvent was distilled off under reduced pressure, and the resulting reaction mixture was subjected to column chromatography using ether/hexane-2.
/3) N-t-zthoxycarbonylallylglycine thiopyridyl ester was obtained as a pale yellow oil. (6
5 (119, yield 91%) Properties: Pale yellow oil NMR spectrum (1 OOMHZ p CDC1a
+ δ): 1.44 (9H, s), 2.54 (2
H, m), 4.48 (I H+ d t , L=
7.0+7.5Hz), 5.0~5.3(3H
, mL 5.70 (IH, aat, , T-18,
0°9.0. 7.0H2), 7.23 (IH,
ddd, J-7, 0°5.0. 2.0H2),,
7.54 (I H, ddd, J-8゜0゜2.
0,1.0Hz), 7.70(IH, add, J-8,
Q.

7.0, 2.0H2), 8.56 (IH, daa,
J-5,0°2.0,1.0Hz).

Reference example 2 N-(t-butoxycarbonyl)-2-amino-4-intenoyl-〇-(t-butyldimethylsilyl)-L-
Threonine thiohyricyl ester N obtained in Example 7
-t-Butoxycarbonylallylglycine-〇-t-Butyldimethylsilyl-L-threonine (in 1,3,1 methylene chloride solution (8 ml), dipyridyl sulfide (
440 mg) then triphenylphosphine (629 m
r) was added and stirred at room temperature for 4 hours. After distilling off the solvent under reduced pressure, it was subjected to column chromatography and ether/
Hexane-2/3) N-t-butoxycarbonylallylglycine-〇-t-butyldimethylsilylthreonine thiopyridyl ester 842m? C yield 82%) was obtained as a pale yellow oil.

Properties: Pale yellow oily substance NMR spectra (60MH2, CDCl3. δ) 0.04 (3H, θ), 0.02 (3H, al
t 0.85 (9H, SL 1.11 (3H, a
, J-7,0Hz).

1.40 (9H, e), 2.58 (2H, m), 4.0
-0-60 (7H1, 66-8.6 (5H2m).

Reference Example 3 (23,3R)-4-(4-t-butyldimethylsilyloxyphenyl)-3-(2-tetrahyto50pyranyl)
Oxy-2-Nt-butoxycarboxylaminobutyric acid-
2-pyridylthiol ester (23,3R)-4-(
4-t-butyldimethylsilyloxyphenyl)-3-(
3 g of 2-tetrahuman (50 pyranyl)oxy-2-Nt-butoxycarbonylaminobutyric H1,0 was dissolved in 10 mJK of anhydrous dichloromethane, and triphenylphosphine 95
4 m9 of dipyridyl sulfide and 712~ of dipyridyl sulfide were added thereto, and the mixture was stirred at 40° C. for 8.5 hours and at room temperature for 14 hours under a nitrogen stream. The reaction solution was concentrated and subjected to silica gel column chromatography and eluted with hexane:ether (1:1) to obtain the title compound 8.
46mf (69% yield) was obtained.

Properties: Oily substance IH film (film, cm): 3450°?
S, x, -? Vector (m/z): 464 NMR spectrometer (CD (J3. δC0,16 (6H, day).

0.96 (9H, θ), 1.54 (9H, s), 6.7
4 and 706 (each 2H, d, J-8H2), 7
．． 24 (IH.

m), 7.44-7.80 (2H, m), 8.52 (I
H.

m).

(Effects of the Invention) By using the method of the present invention, oputide can be synthesized. This method is characterized by the use of an organic solvent, and it is thought that theoretically any polymeric peptide can be synthesized as long as there is a solvent that can solubilize it. The peptides obtained by the method of the present invention are basic substances that can be applied in a wide range of fields such as hormones, agricultural chemicals, and foods, so the method of the present invention that can efficiently produce the peptides will be of great benefit to this field. Dew.

(5 people outside)

Claims (7)

[Claims] (1) General formula (1) ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (1) [In the formula, R^1 is a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, phenyl group, carbon number 7
~9 aralkyl group, a C1-6 alkyl group substituted with a protected or unprotected hydroxyl group, a C7-9 aralkyl group substituted with a protected or unprotected hydroxyl group,
Represents an alkyl group having 2 to 6 carbon atoms substituted with a lower alkylthio group, an alkyl group having 1 to 4 carbon atoms substituted with a protected or unprotected thiol group, or an indolylmethyl group, R^2 is a hydrogen atom, Protecting group or group for amino group ▲ Formula,
There are chemical formulas, tables, etc. ▼ (where R^1 represents the same meaning as above, R^4 represents a hydrogen atom or a protecting group for an amino group), and R^3 represents a hydrogen atom or a lower alkyl group. A method for producing a peptide having the general formula (2) ▲ Numerical formula, chemical formula, table, etc. ▼ (2) (wherein R^5 is a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, Alkenyl group having 2 to 6 carbon atoms, phenyl group, 7 carbon atoms
~9 aralkyl group, a C1-6 alkyl group substituted with a protected or unprotected hydroxyl group, a C7-9 aralkyl group substituted with a protected or unprotected hydroxyl group,
Alkyl group having 2 to 6 carbon atoms substituted with lower alkylthio group, 1 to 4 carbon atoms substituted with protected thiol group
An α-amino acid having an alkyl group or an indolylmethyl group) is reacted with trialkylsilylimidazole, and then the general formula (3) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(3) [In the formula, R^6 is hydrogen atom, alkyl group having 1 to 10 carbon atoms, alkenyl group having 2 to 6 carbon atoms, phenyl group, 7 carbon atoms
-9 aralkyl group, alkyl group having 1 to 6 carbon atoms substituted with a protected hydroxyl group, aralkyl group having 7 to 9 carbon atoms substituted with a protected hydroxyl group, carbon substituted with a lower alkylthio group Represents an alkyl group of 2 to 6 carbon atoms, an alkyl group of 1 to 4 carbon atoms substituted with a protected thiol group, or an indolylmethyl group, R^7 is a protecting group or group for an amino group ▲ Numerical formula, chemical formula, table etc. ▼ (Here, R^6 represents the same meaning as above, and R^8 represents a protecting group for the amino group) An active ester compound having the following is reacted in an organic solvent, or deprotected after the reaction. or a method characterized by alkylation after the reaction. (2) The method according to claim 1, wherein the trialkylsilylimidazole is t-butyldimethylsilylimidazole, trimethylsilylimidazole, triethylsilylimidazole, dimethylphenylsilylimidazole, or t-butyldiphenylsilylimidazole. (3) The method according to claim 1, wherein the solvent used in the peptide formation reaction is an amide solvent, an ether solvent, or a chlorine solvent. (4) The reaction temperature in the peptide formation method is 30 to 0°C
The method according to claim 1. (5) The method according to claim 1, wherein the amount of trialkylsilylimidazole used in the reaction is 1 to 2 equivalents relative to the α-amino acid group of formula (2). (6) The method according to claim 1, wherein the formulas (2) and (3) are optically active substances. (7) The method according to claim 1, wherein the formula (1) is an optically active substance.